The proposed research centers around the molecular biology of osmoregulation n two very different bacteria, Salmonella typhimurium and Rhizobium meliloti. Osmoregulation is a fundamental problem confronting all cells. A better understanding of how bacteria cope with osmotic stress would provide valuable insight for all cells. We find that when S. typhimurium is shifted to medium of high osmolarity, approximately 20% of the proteins in the cells is lost. We would like to determine if this protein is periplasmic protein or if a fraction of diverse cytoplasmic protein is lost. We will also investigate the situation when cells grow in artificial urine to determine if the bacterium is osmotically compromised. We would determine if osmoprotectants like proline and betaine affect this. For many bacteria but not the R. meliloti, the amino acid proline is an osmoprotectant. It is possible that the bacterium degrades proline using proline oxidase before it can accumulate. Mutants lacking the activity will be isolated and tested. Proline transport could be limited by osmotic stress. This will also be tested. A simple bio-assay for proline in bacterial cells will be developed. It will be seen if S. typhimurium known to be protected by proline accumulates appreciable quantities. We will also test all sorts of other bacteria growing in partially defined medium with osmotic stress to determine how common this is. The proposed research provides a variety of physiological, genetic, and biochemical problems. If adequate funding is available, the research can also include recombinant DNA research to clone the genes for proline oxidase. It is designed to be carried out by undergraduates. But the first two aspects can be expanded to be appropriate for graduate degrees.